Recognition and separation of carbon clusters

ABSTRACT

Carbon clusters, such as C 60  and C 70  fullerenes are separated by means of a recognition selector having the formula: ##STR1## wherein R 1  is ##STR2## wherein R 2  is O, S or NR 12  wherein R 12  is independently hydrogen or P═O with the proviso that when R 12  is P═O, then only one such group is present and all R 2  &#39;s are additionally bonded to R 12 , 
     R 4  is independently O, S or NH, 
     R 3  and R 5  are each independently hydrogen or lower alkyl, 
     n and o are each independently zero, 1, 2 or 3, 
     p, q, r, s and t are each independently zero or 1, 
     Ar is a monocyclic or ortho-fused polycyclic aromatic moiety having up to 10 carbon atoms, either of which may be unsubstituted or substituted with one or more lower alkyl, NO 2 , N(R 6 ) 3   + , CN, COOR 7 , SO 3  H, COR 8  and OR 9  wherein R 6 , R 7 , R 8  and R 9  are each independently hydrogen or lower alkyl; 
     W is H or CH═CH 2  ; and 
     m is 1 to 10.

The invention described herein was made with Government support underGrant NSF CHE 87-14950 awarded by The National Science Foundation. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the recognition and separation ofcarbon clusters. In one aspect, the invention is directed to arecognition selector useful, for example, as a stationary phase in thechromatographic separation of carbon clusters, such as C₆₀ and C₇₀fullerenes. In another aspect, the invention is directed to the use ofelevated temperatures to obtain improved separation of carbon clusters,such as C₆₀ and C₇₀ fullerenes.

2. Description of the Prior Art

Solid, or elemental, carbon has for some time been thought to exist inonly two forms: diamond, wherein the structure is face-centered cubicwith each carbon atom bonded to four other carbon atoms in the form of atetrahedron: and graphite, which has layers of carbon atoms arranged inthe form of hexagons lying in planes, the carbon atoms of various layersbeing aligned with each other.

Recently, a third form of solid carbon has been discovered. Unlikediamond and graphite, however, this new form of carbon has the structureof a closed shell. This family of closed carbon shells, also referred toherein as carbon clusters, include for example that group of closedcarbon shells denominated in the art as the fullerenes, such as thosehighly stable molecules known as Buckminsterfullerene, and the relatedmolecule known as fullerene-70; other members of the fullerene familyinclude, e.g., C₇₈ and C₇₈ and C₂₄₀.

Buckminsterfullerene, also known as C₆₀, is a 60 carbon atom moleculehaving the geometry of a truncated icosahedron; that is, a polygon with60 vertices whereat the carbon atoms are placed, and 32 faces, 12 ofwhich are pentagons and 20 of which are hexagons. C₆₀ thus has thegeometry of a soccerball.

Fullerene-70, also known as C₇₀, is similar to C₆₀, only it has 10additional carbon atoms which are believed to be inserted as a band ofhexagons around the middle of the truncated icosahedron.

Although only recently discovered, various routes for synthesizingfullerenes are already available. An aspect of fullerene production thatis of especial importance is the separation of fullerenes, as a whole,from a resultant fullerene-containing product admixture, as well as theseparation of the various species of fullerenes from each other.

Presently known separation techniques include the method described byTaylor, et al. in J. Chem. Soc., Chem. Commun., pp. 1423-1425 (1990).This method employs solvent extraction to remove C₆₀ and C₇₀ from carbonproduct deposits, followed by chromatographic separation using aluminaand hexane to separate C₆₀ from C₇₀.

Another approach, which is reported by Hawkins, et al. in J. Org. Chem.,55, pp. 6250-6252 (1990) entails the use of flash chromatography by dryloading onto silica gel, and elution with hexanes to achieve a 40%recovery of material that is almost exclusively C₆₀ and C₇₀. C₆₀ and C₇₀are then separated chromatographically using a commercially availableN-3,5-(dinitrobenzoyl)-phenylglycine derived stationary phase elutedwith hexane. Cox, et al. have also described the chromatographicpurification of C₆₀ and C₇₀ using a π-acidic dinitroaniline stationaryphase in J. Amer. Chem. Soc., 113, pp. 2940 (1991).

Notwithstanding these developments, the methods for separating andpurifying fullerenes, as known heretofore, have been only marginallysuccessful insofar as practical analytic and, more importantly,preparative scale applications are concerned. For example, thechromatographic separations that are described by Taylor, et al. andHawkins, et al. are impractical for preparative scale preparation offullerenes because of the extreme insolubility of the fullerene analytesin the mobile phase. This condition limits, for example, the samplesizes which can be separated per run.

Thus there continues to be a pressing need for a method of separatingfullerenes in a manner that is qualitatively and quantitatively superiorto known methods, and that will be of practical utility in both theanalytic and preparative scale separation of carbon clusters, such asC₆₀ and C₇₀ fullerenes.

SUMMARY OF THE INVENTION

The present invention overcomes the inadequacies attendant carboncluster separation methods known hitherto. In one aspect, the presentinvention is directed to a recognition selector having the formuladescribed hereinbelow, which formula provides multiple π-acidic sitesarranged in a concave-like shape that is complementary to theconvex-like surface shape of a carbon cluster molecule. The recognitionselector of the invention is thus capable of undergoing simultaneousmultipoint interactions with carbon cluster molecules, particularly C₆₀and C₇₀ fullerenes.

The recognition selector of the invention is a compound having theformula: ##STR3## wherein R₁ is ##STR4## and wherein R₂ is O, S or NR₁₂wherein R₁₂ is independently hydrogen or P═O with the proviso that whenR₁₂ is P═O, then only one such group is present and all R₂ 's areadditionally bonded to R₁₂ ; R₄ is independently O, S or NH; R₃ and R₅are each independently hydrogen or lower alkyl; n and o are eachindependently zero, 1, 2 or 3; p, q, r, s and t are each independentlyzero or 1 and Ar is a monocyclic or ortho-fused polycyclic aromaticmoiety having up to 10 carbon atoms, either of which may beunsubstituted or substituted with one or more lower alkyl, NO₂, N(R₆)₃⁺, CN, COOR₇, SO₃ H, COR₈ and OR₉ wherein R₆, R₇, R₈ and R₉ are eachindependently hydrogen or lower alkyl; W is H or CH═CH_(2;) and m is 1to 10.

In an embodiment of the present invention, the above-describedrecognition selector is employed in a method for separating a carboncluster, such as a fullerene, from a mixture containing at least onecarbon cluster which comprises contacting a mixture containing at leastone carbon cluster with the recognition selector described above, underconditions effective to form a complex between said carbon cluster andsaid recognition selector, separating said complex from said mixture andrecovering said carbon cluster from said complex.

In another embodiment, the present invention is also directed to anapparatus, such as a liquid membrane separation system or a liquidchromatographic column, such as a high performance liquidchromatographic (HPLC) column, that employs the recognition selectordescribed above.

In still another embodiment, the present invention is directed to theuse of elevated temperatures to obtain improved chromatographicseparation of carbon clusters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the affect of temperature on the retentionof C₆₀, C₇₀ and anthracene.

FIG. 2A is a graph showing the preparative separation of C₆₀ and C₇₀ atroom temperature.

FIG. 2B is a graph showing the preparative separation of C₆₀ and C₇₀ at90° C.

FIG. 3 is a graph showing the relative capacity factors for tendifferent analytes, including C₆₀ and C₇₀, using a π-acidicdinitrobenzoate ester stationary phase, SP 8, and a stationary phaseformed with a recognition selector (RS 3) of the present invention.

FIG. 4 is a graph showing the relative capacity factors for tendifferent analytes, including C₆₀ and C₇₀, using stationary phasesformed with recognition selectors RS 3 and RS 7 of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for recognizing andseparating carbon clusters, particularly those compounds known asfullerenes and more particularly those known as C₆₀ to C₇₀. The methodemploys a recognition selector compound having the formula describedbelow. Significantly, in the practice of the invention, a qualitativeand quantitative increase in the separation of fullerenes is obtained ascompared to methods previously known.

The recognition selector of the present invention is a compound havingthe following formula: ##STR5## wherein R₁ is ##STR6## wherein R₂ is O,S or NR₁₂ wherein R₁₂ is independently hydrogen or P═O with the provisothat when R₁₂ is P═O, then only one such group is present and all R₂ 'sare additionally bonded to R₁₂ ;

R₄ is independently O, S or NH,

R₃ and R₅ are each independently hydrogen or lower alkyl,

n and o are each independently zero, 1, 2 or 3,

p, q, r, s and t are each independently zero or 1,

Ar is a monocyclic or ortho-fused polycyclic aromatic moiety having upto 10 carbon atoms, either of which may be unsubstituted or substitutedwith one or more lower alkyl, NO₂, N(R₆)₃ ⁺, CN, COOR₇, SO₃ H, COR₈ andOR₉ groups wherein R₆, R₇, R₈ and R₉ are each independently hydrogen orlower alkyl;

W is H or CH═CH_(2;) and

m is 1 to 10.

As employed herein, the lower alkyl groups contain up to 6 carbon atomswhich may be in the normal or branched configuration, including methyl,ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, amyl, pentyl, hexyland the like. The preferred alkyl group contains 1 to 3 carbon atoms;methyl is particularly preferred.

The monocyclic or ortho-fused polycyclic aromatic moiety having up to 10carbon atoms includes, e.g., phenyl, α-naphthyl and β-naphthyl.Preferred substituted monocyclic aromatic moieties include3,5-dinitrophenyl and 2,4-dinitrophenyl. Preferred substitutedpolycyclic aromatic moleties include 6-methoxy-β-naphthyl and7-methoxy-β-naphthyl.

In a first preferred embodiment of the recognition selector of thepresent invention, n and p are each 1; o, q, r, s and t are each zero;R₂ is O; Ar is 3,5-dinitrophenyl; m is 7; and W is CH═CH₂. Therecognition selector of this first preferred embodiment is denotedhereinafter as RS 3. The structure of RS 3 is shown below. ##STR7##

In a second preferred embodiment of the recognition selector of thepresent invention, n and p are each 1; o, q, r, s and t are each zero;R₂ is NH; Ar is 3,5-dinitrophenyl; m is 7; and W is CH═CH₂. Therecognition selector of this second preferred embodiment is denotedhereinafter as RS 5. The structure of RS 5 is shown below. ##STR8##

In a third preferred embodiment of the recognition selector of thepresent invention, n, o, p and r are each 1; q, s and t are each zero;R₂ is O; R₄ is NH; Ar is 3,5-dinitrophenyl; m is 7; and W is CH═CH₂. Therecognition selector of this third embodiment is denoted hereinafter asRS 6. The structure of RS 6 is shown below. ##STR9##

In a fourth preferred embodiment of the recognition selector of thepresent invention n is 1; o, p, q, r, s and t are each zero; R₂ is O, Aris 2,4-dinitrophenyl; m is 7; and W is CH═CH₂. The recognition selectorof this fourth embodiment is denoted hereinafter as RS 7. The structureof RS 7 is shown below. ##STR10##

In a fifth preferred embodiment of the recognition selector of thepresent invention, n, p, q, r and s are each 1; o and t are each zero;R₂ and R₄ are each NH; R₃ is isobutyl; Ar is 3,5-dinitrophenyl; m is 7and W is CH═CH₂. The recognition selector of this fifth embodiment isdenoted hereinafter as RS 8. ##STR11##

In a sixth preferred embodiment of the recognition selector of thepresent invention, n, p, q, and r are each 1; o, s and t are each zero;R₂ is O; R₃ is CH₃ ; R₄ is NH, Ar is β-naphthyl; m is 6; and W isCH═CH₂. The recognition selector of this sixth embodiment is denotedhereinafter as RS 9. The structure of RS 9 is shown below. ##STR12##

In a seventh preferred embodiment of the recognition selector of thepresent invention, n, p and q are each 1; o, r, s and t are each zero;R₂ is O; R₃ is CH₃ ; Ar is 6-methoxy-β-naphthyl; m is 7 and W is CH═CH₂.The recognition selector of this seventh embodiment is denotedhereinafter as RS 10. The structure of RS 10 is shown below. ##STR13##

In an eighth preferred embodiment of the recognition selector of thepresent invention, n, o, p, r and t are each 1; q and s are each zero;R₂ is O; R₄ is NH; R₅ is CH₃ ; Ar is α-naphthyl; m is 7; and W isCH═CH₂. The recognition selector of this eighth embodiment is denotedhereinafter as RS 11. The structure of RS 11 is shown below. ##STR14##

In a ninth preferred embodiment of the recognition selector of thepresent invention, n and q are each 1; o, p, r, s and t are each zero;R₂ is NR₁₂ and R₁₂ is P═O with the proviso that only one such P═O groupis present and all R₂ 's are additionally bonded to said P═O group; Aris α-naphthyl; m is 7 and W is CH═CH₂. The recognition selector of thisninth embodiment is denoted hereinafter as RS 12. The structure of RS 12is shown below. ##STR15##

The recognition selectors of the present invention may be prepared byconventional chemical preparative techniques. The recognition selectorsof the present invention may be utilized to form a stationary phase ofan HPLC column by techniques known in the art. A preferred technique inthis regard includes hydrosilation followed by immobilization on asupport effective for use in chromatographic separation, such as silica(denoted herein as SiO₂) or alumina. For illustrative purposes thepreparation of recognition selectors RS 3 and RS 7 are described below,but one skilled in the art will readily appreciate the modificationsnecessary to prepare other recognition selectors within the scope of thechemical formula that is described herein.

The synthetic sequence used to prepare RS 3 is shown below in Table 1.

                                      TABLE 1                                     __________________________________________________________________________     ##STR16##                                                                     ##STR17##                                                                     ##STR18##                                                                    __________________________________________________________________________

This preparation begins with an aldehyde, ω-undecenylenyl aldehyde (A).This aldehyde has a terminal double bond which serves as a means forattachment to silica to form a stationary phase in an HPLC column.Treatment of the aldehyde with formaldehyde under basic conditions usingpotassium hydroxide, ethanol and water, provides the triol (B). Thetriol is acylated with 3,5-dinitrobenzoyl chloride in triethylamine anddichloromethane to afford the triester, RS 3. Hydrosilation usingdimethylchlorosilane, chloroplatinic acid (cat) and dichloromethane,followed by treatment with ethanol, triethylamine and ethyl etheraffords the silane (C). Immobilization on silica gel (5μ/100 Å silicagel, 120° C., 1 torr, 24 hr) affords stationary phase RSP 3.

The synthetic sequence used to prepare RS 7 is shown below in Table 2.

                  TABLE 2                                                         ______________________________________                                         ##STR19##                                                                     ##STR20##                                                                     ##STR21##                                                                    ______________________________________                                    

The triol (B) prepared as described above, is reacted with2,4-dinitrofluorobenzene in triethylamine and dichloromethane to yieldthe triether, RS 7. Hydrosilation of triether RS 7 withdimethylchlorsilane, chloroplatinic acid (cat) and dichloromethane,followed by treatment with ethanol, triethylamine and ethyl etheraffords the silane, D. Bonding of the silane to silica gel (5μ/100 Åsilica gel, 120° C., 1 torr, 24 hr) affords stationary phase RSP 7.

Separation of carbon clusters such as fullerenes using the recognitionselector of the invention may be achieved in a variety of techniquesknown in the art. In one embodiment, the recognition selector may formthe active portion of the stationary phase in an HPLC column asdescribed above. In this embodiment of the present invention, theterminal W of the formula is preferably CH═CH₂ so as to permit thechiral selector to be immobilized on a support which is suitable for usein chromatographic separations. Supports in this regard include, e.g.,silica and alumina. In one configuration, the recognition selector isimmobilized by covalently bonding it to silica. Those recognitionselectors of the instant invention that are optically active, may, ifdesired, be separated into the R or S enantiomer for use as the activeportion of the stationary phase in the column and may thus also beemployed in enantiomeric separations.

The effect of temperature on the chromatographic behavior of fullerenesis unusual. Generally, in chromatographic separations, the thermodynamicparameters of adsorption indicate that a loss of entropy accompaniesexothermic adsorption. Thus, as a rule, an increase in chromatographiccolumn temperature translates into a lessening of analyte retention. Itis in this regard that the behavior of fullerenes is unusual; that is,the retention of fullerenes such as C₆₀ and C₇₀ increases rather thandecreases when column temperature is increased. This unusual temperaturedependence of fullerene retention has been observed to occur in avariety of mobile phases and with several different π-acidic and π-basicstationary phases. Since polynuclear aromatic dopants, such asanthracene and naphthalene, exhibit normal retention behavior under thesame conditions, the unusual temperature effect relative to fullerenesseems to be analyte dependent and not column or mobile phase dependent.

Fullerenes, such as C₆₀ and C₇₀, are, in the normal course ofchromatographic separation, present in solution with, e.g., benzene.Thus an increase in column temperature leads to markedly improvedbandshapes and resolution of C₇₀ and C₇₀ : the higher column temperatureleads to an increase in the retention of the fullerenes whilesimultaneously causing a decrease in retention time of the solvent.Improved separation of fullerenes using this unusual temperature effectcan be obtained on chromatographic columns utilizing a stationary phaseformed from the recognition selector of the invention, as well as oncommercially available columns. Examples of commercially availablecolumns include those whose stationary phases incorporate as an activepart, 3,5-dinitrobenzoyl leucine, 3,5-dinitrobenzoyl phenylglycine,naphthylalanine and naphthyl leucine. These columns are commerciallyavailable from Regis Chemical Company, Morton Grove, Ill.

An example of the effect of temperature on the retention of C₆₀ and C₇₀is shown in FIG. 1. As shown in FIG. 1, a commercially available columnobtained from Regis Chemical Company employing a stationary phase of(R)-N-(3,5-dinitrobenzoyl)phenylglycine (4.6 mm I.D.×25 cm length) wasused to study the effect of temperature on C₆₀, C₇₀ and anthracene. Allsamples were dissolved in a mobile phase of 10% dichloromethane inhexane prior to injection. Flow rate was 2.00 ml/min. and void time wasdetermined by injection of 1,3,5-tri-t-butylbenzene, as described byPirkle, et al. in J. Liq. Chrom., 14, 1, 1991 the contents of which areincorporated herein by reference. As seen by FIG. 1, as columntemperature increased, the retention of C₆₀ and C₇₀ increased; whereasthe retention of anthracene decreased.

The practical effect of this unusual temperature dependence is shown inFIGS. 2A and 2B. The column utilized to generate the preparativeseparations shown in FIGS. 2A and 2B was obtained from Regis ChemicalCompany and employed a (R)-3,5-(dinitrobenzoyl)phenylglycine (21.1 mmI.D.×25 cm length) as a stationary phase. Flow rate was 9 ml/min.;mobile phase was hexane. The sample utilized in each case was a 500 μlinjection of a 6 mg/ml benzene solution of C₆₀ and C₇₀. A comparison ofFIG. 2A, wherein separation was carried out at room temperature (about23° C.), and FIG. 2B, wherein separation was carried out at 90° C.,shows an improvement in bandshape and resolution when the highertemperature is used.

The increased column temperatures contemplated by the present inventionare temperatures that are higher than room temperature, preferably inthe range from over about room temperature (about 25° C.) to about 120°C. More preferably, the increased temperature range is between about 80°C. to about 100° C. Most preferably, column temperature is about 90° C.

In another embodiment, the recognition selector of the invention may beutilized to effect separations employing semi-permeable membraneswherein the recognition selector forms part of a mobile phase. Suchtechniques include the use of semi-permeable membranes that are in theform of hollow fiber membranes. In this embodiment of the invention, itis preferred that the terminal W in the formula of the recognitionselector be hydrogen so as to minimize covalent bonding by therecognition selector. In one particularly useful embodiment, therecognition selector forms part of a liquid membrane passing on one sideof a semi-permeable barrier with the fullerenes to be separated passingon the other side of the barrier. The pores of the barrier becomeimpregnated with the liquid membrane containing the recognitionselector. A fullerene or species of fullerene complexes with therecognition selector, passes through the barrier into the moving liquidmembrane and is conducted to a second location where dissociation of thecomplex takes place thus allowing the fullerene to be recovered. Thistechnique is generally disclosed in commonly assigned U.S. patentapplication Ser. No. 528,007, filed May 23, 1990, now U.S. Pat. No.5,080,795 the contents of which are incorporated herein by reference.

The following examples are given to illustrate the scope of theinvention. Because these examples are given for illustrative purposesonly, the invention embodied therein should not be limited thereto.

EXAMPLES

A series of experiments was conducted to examine the chromatographicbehavior of C₆₀ and C₇₀ and eight polycyclic aromatic hydrocarbons usingstationary phases formed from the recognition selector of the presentinvention, and to compare this behavior with commercially availablestationary phases, as well as four different π-acidic stationary phasesthat were especially prepared for purposes of these experiments.

The polycyclic aromatic hydrocarbons and the fullerenes that were usedin these studies are shown in Table 3, below.

                                      TABLE 3                                     __________________________________________________________________________     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                    __________________________________________________________________________

HPLC columns having four different commercially available stationaryphases were obtained from Regis Chemical Company, Morton Grove, Ill.These stationary phases, denominated for experimental purposes as SP 1,SP 2, SP 3 and SP 4, are depicted in Table 4, below.

                                      TABLE 4                                     __________________________________________________________________________     ##STR25##                                         SP 1                        ##STR26##                                         SP 2                        ##STR27##                                         SP 3                        ##STR28##                                         SP 4                       __________________________________________________________________________

APPARATUS

Chromatographic analysis was performed using a Beckman-Altex 100-A pump,a Rheodyne Model 7125 injector with a 20 μl sample loop, a linear UVIS200 variable wavelength absorbance monitor set at 254 nm, and theHewlett-Packard HP 3394-A integrating recorder.

MATERIALS

Rexchrom™ 5μ/100 Å silica gel and columns containing stationary phasesSP 1-4 were obtained from Regis Chemical Company, Morton Grove, Ill.Dimethylchlorosilane and 4-aminobutyldimethylmethoxysilane were obtainedfrom Petrarch Systems, Bristol, Pa.

METHODS

All chromatographic experiments were carried out at a nominal flow rateof 2.00 ml/min. Column void time was determined by injection oftri-t-butylbenzene in the manner reported by Pirkle, et al., in J. Liq.Chrom., 14, pp. 1-8, 1991 the contents of which are incorporated hereinby reference.

SYNTHESIS

The four π-acidic stationary phases, SP 5, SP 6, SP 7 and SP 8, thatwere prepared for comparative purposes and immobilized on silica areshown in Table 5, below.

                                      TABLE 5                                     __________________________________________________________________________     ##STR29##                                         SP-5                        ##STR30##                                         SP 6                        ##STR31##                                         SP 7                        ##STR32##                                         SP 8                       __________________________________________________________________________

PREPARATION OF SP 5

The synthetic route for SP 5, which was an achiral glycine analog isshown below: ##STR33##

Preparation of 3,5-(Dinitrobenzamido)glycine, (2): Glycine ((1), 5.0 g)was suspended in 100 ml dry tetrahydrofuran (THF) and cooled in an icebath. 3,5-dinitrobenzoyl chloride (16.9 g) and propylene oxide (7.0 ml)were added, and the mixture was stirred under a nitrogen atmospherewhile gradually warming to room temperature. After 10 h the crudereaction mixture was evaporated to afford a brown oil. Addition of 100ml of dichloromethane resulted in crystallization after several minutes.Filtration followed by several washes with dichloromethane and dryingunder high vacuum gave 13.9 g of 3,5-(Dinitrobenzamido)glycine, (2),(78% yield) as a pale yellow powder. ¹ H NMR (d₆ DMSO)δ: 12.8 (s,1H),9.6(t,1H), 9.1 (s,2H), 9.0 (s,1H), 4.0 (d,2H).

Preparation of the Organosilane (3): To a cooled (ice bath) solution of1.0 g of 3,5-(Dinitrobenzamido)glycine, (2), in tetrahydrofuran wasadded 0.92 g of 1-ethoxy-carbonyl-2-ethoxyl-1,2-dihydroquinoline (EEDQ).After stirring 45 minutes under a nitrogen atmosphere, 0.50 g of4-aminobutyldimethylmethoxysilane was added and the reaction mixture wasallowed to gradually warm to room temperature. After 10 h the crudereaction mixture was evaporated and purified by flash chromatography onsilica to give 0.65 g (42% yield) of silanized3,5-(Dinitrobenzamido)glycine, (3), as a slightly pink foam.

Silica gel (5.0 g, Rexchrom™, 5μ/100 Å) was placed in a round bottomflask fitted with a Dean-Stark trap, condenser, and boiling stick. About30 ml of benzene were added, and the mixture was refluxed for severalhours. Dimethylformamide (1 mL) was then added to the benzene slurry,and the sample was evaporated to near dryness on a rotary evaporator. Adichloromethane solution of silanized 3,5-(Dinitrobenzamido)glycine,(3), (0.65 g) was then added, and the resulting slurry was sonicated forseveral minutes before being evaporated to near dryness. The sample wasagain slurried in dichloromethane, sonicated, and evaporated to neardryness, this sequence being repeated several times to insure completecoverage of the silica gel. The nearly dry silica gel-silane compound(3) mixture was then heated on an oil bath under reduced pressure (130°C., 1 mm Hg, 18 hours). The silica gel was then slurried in ethanol,filtered through a fine sintered glass funnel, and washed repeatedlywith ethanol, and then methanol. Evaporation and analysis of the ethanolwashes could have been performed at this point to look for degradationof the silane compound (3) during the course of the bonding reaction.The washed silica gel was then slurried in methanol and packed into a4.6 mm I.D.×25 cm length stainless steel HPLC column using an air drivenHaskell pump operating at about 9000 psi. Recovered excess stationaryphase from the column packer was dried thoroughly under high vacuum andsubmitted for elemental analysis (C 3.39%) which indicated a loading of1.9×10⁻⁴ moles of selector per gram of stationary phase. Residualsilanols on the chromatographic support were then a "endcapped" bypassing a solution of 1 ml hexamethyldisilazane dissolved in 50 mldichloromethane through the dichloromethane-equilibrated column at aflow rate of 1 ml/min. The column was then sequentially eluted withdichloromethane, methanol, and 20% 2-propanol in hexane.

PREPARATION OF SP 6

The synthetic route for SP 6, which contained an isolated3,5-dinitrobenzamide system, is shown below. ##STR34##

Preparation of the Organosilane, (5): Triethylamine (0.69 g) and4-aminobutyldimethylmethoxysilane, (4), (1.00 g) were dissolved in 20 mLdichloromethane and cooled in an ice bath. 3,5-dinitrobenzoyl chloride(1.43 g) dissolved in 10 ml of dichloromethane, was then added dropwiseover several minutes with stirring under a nitrogen atmosphere. Thereaction mixture was allowed to warm to room temperature and stirred foran additional hour. The crude reaction mixture was evaporated to drynessand purified by flash chromatography (silica gel, 5%acetonitrile/dichloromethane) to afford the organosilane (5) as a clearoil (840 mg, 69.4% yield). ¹ H NMR (CDCl₃)δ: 9.15 (s,1H), 9.0 (s,1H),6.8 (bs,1H), 3.55 (m,2H), 4.3 (s,3H), 1.75 (m,2H), 1.5 (m,2H), 0.65(t,2H), 0.15 (s,6H).

Bonding of the organosilane (5) to silica, and packing of the resultingstationary phase into an HPLC column followed the procedure reported forthe preparation of stationary phase SP 5, except that a Kugelrohrdistillation apparatus was used in the bonding reaction (130° C., 1 mmHg, 18 hours). Stationary phase recovered from the column packer wassubmitted for elemental analysis (C 4.69%) which indicated a loading of3.0×10⁻⁴ moles of selector per gram of stationary phase.

PREPARATION OF SP 7

The synthetic route for SP 7, which contained a 2,4-dinitroanilinesystem, is shown below: ##STR35##

Preparation of the Olefin, (7): To a cooled, stirred solution ofallylamine ((6), 0.46 g) and triethylamine (0.90 g) in dichloromethane,was added 2,4-dinitrofluorobenzene (1.5 g). After warming to roomtemperature, the reaction mixture was stirred for three hours, thenevaporated to dryness and purified by flash chromatography (silica,dichloromethane) to afford olefin (7) as a crystalline solid (1.09 g,61% yield). ¹ H NMR (CDCl₃)δ: 9.25 (d,1H), 8.8 (bs,1H), 8.3 (dd,1H), 7.0(d,1H), 6.0 (m,1H), 5.8 (dd,2H), 4.1 (t,2H).

Preparation of the Organosilane (8): Olefin (7) (1.09 g) was dissolvedin 10 ml dichloromethane and 10 ml dimethylchlorosilane. Chloroplatinicacid (10 mg) dissolved in a minimum of 2-propanol was then added, andthe mixture was heated at reflux. Progress of the reaction was monitoredby disappearance of starting material in quenched reaction aliquots.(quenching solution was composed of 5 ml absolute ethanol, 5 mltriethylamine and 5 ml diethyl ether). The assay procedure consisted ofremoving several drops of reaction mixture, evaporating to dryness underhigh vacuum to remove excess dimethylchlorosilane and adding severaldrops of quenching solution. The mixture was then heated for severalminutes on an oil bath, diluted with dichloromethane, and examined byTLC. After about three hours, TLC analysis of quenched reaction aliquotsindicated complete consumption of starting material. The crude reactionmixture was evaporated and purified by flash chromatography on silicausing 2% ethanol in dichloromethane as eluent to afford 0.80 g oforganosilane (8) (50% yield). ¹ H NMR (CDCl₃)δ: 9.2 (s,1H), 8.6 (bs,1H),8.3 (d,1H), 7.0 (d,1H), 3.6 (m,2H), 3.4 (m,2H), 1.8 (m,2H), 1.2 (t,3H),0.7 (t,2H), 0.1 (s,6H).

Bonding of the organosilane (8), and packing of the resulting stationaryphase into an HPLC column followed the procedure reported for thepreparation of SP 5, except that a Kugelrohr distillation apparatus wasused in the bonding reaction (115° C., 1 mm Hg, 24 hours). Stationaryphase recovered from the column packer was submitted for elementalanalysis (C, 3.28%) which indicated a loading of 2.5×10⁻⁴ of selectorper gram of stationary phase.

PREPARATION OF SP 8

The synthetic route for SP 8, which contained a 3,5-dinitrobenzoateester, is shown below. ##STR36##

Preparation of the Olefin (10): To a cooled solution of ω-undecenylenylalcohol ((9), 1.0 g) and triethylamine (0.65 g) in 10 mL drytetrahydrofuran, was added 3,5-dinitrobenzoyl chloride (1.36 g) withstirring. The reaction mixture was allowed to warm to room temperature,and was then stirred for one hour. The heterogeneous solution was thendiluted with ether and extracted three times with a 1.0M HCl solution.The organic layer was washed with water, then brine, then dried overanhydrous magnesium sulfate. Filtration and evaporation yielded theolefin (10) (2.00 g, 93% yield) as a pale yellow solid. ¹ H NMR(CDCl₃)δ: 9.25 (d,1H), 9.2 (d,2H), 5.8 (m,1H), 4.95 (m,2H), 4.4 (t,2H),2.05 (m,2H), 1.85 (m,2H), 0.9 (m,12H).

Preparation of the Organosilane (11): The olefin (10) was converted intoethoxysilane (11) using the hydrosilylation procedure reported forpreparation of organosilane (8). Crude ethoxysilane (11) was purified byflash chromatography on silica using dichloromethane as eluent to afford1.04 g of ethoxysilane (11) (80.6% yield) as a yellow oil. ¹ H NMR(CDCl₃)δ: 9.25 (d,1H), 9.15 (d,2H), 4.45 (t,2H), 3.65 (q,2H), 1.85(m,2H), 1.3 (m,16H), 0.6 (t,2H), 0.1 (s,6H).

Bonding of ethoxysilane (11) to silica, and packing of the resultingstationary phase into an HPLC column followed the procedure reported forthe preparation of stationary phase SP 5 (120° C., 1 mm Hg, 24 hours).Stationary phase recovered from the column packer was submitted forelemental analysis (C, 3.94%) indicated a loading of 1.6×10⁻⁴ moles/g.

PREPARATION OF RECOGNITION SELECTOR RS 3

The synthetic route for RS 3 is shown in Table 1 supra.

Preparation of the Triol (B): Undecylenic aldehyde (A) (50 g) and 200 g40% formaldehyde solution were dissolved in 500 ml of a 1:1 mixture ofabsolute ethanol and water. Potassium hydroxide (16.30 g) dissolved in150 ml of a 1:1 ethanol/water mixture was then added dropwise to thestirring solution at 0° C. The reaction was allowed to warm to roomtemperature, stirred for four hours, then heated to 60° C. and stirredfor an additional two hours at which time TLC indicated completeconsumption of starting material and formation of a new product spot.The crude reaction mixture was concentrated under vacuum, then extractedseveral times with ether. The combined ether extracts were then washedseveral times with water, washed with brine, dried over anhydrousmagnesium sulfate, filtered and evaporated to dryness. Flashchromatography on silica gel using 10% methanol in dichloromethane aseluent gave triol (A) (28.2 g, 41.4% yield) as a white solid. ¹ H NMR(CDCl₃)δ: 5.85 (m,1H), 5.0 (m,2H), 3.75 (d,6H), 2.75 (t,3H), 2.05(m,2H), 1.3 (m,12H).

Triol (B) (1.0 g) and triethylamine (1.5 g) were dissolved in 50 ml drytetrahydrofuran and cooled in an ice bath. 3,5-dinitrobenzoyl chloride(3.0 g) was then added, and the solution was allowed to gradually warmto room temperature while stirring overnight under a nitrogenatmosphere. Precipitated triethylammonium hydrochloride was then removedby filtration, and the filtrate was washed several times with a 1M HClsolution, dried over anhydrous magnesium sulfate, filtered, andevaporated to dryness. Ether trituration of the resulting oil gave therecognition selector of the present invention, RS 3 (1.64 g, 47% yield)as a white solid. ¹ H NMR (CDCl₃, d₆ DMSO)δ: 9.2 (d,3H), 9.1 (d,6H),5.75 (m,1H), 4.9 (m,2H), 4.65 (2,6H), 2.0 (m,2H), 1.8 (m,2H), 1.4(m,10H).

Preparation of the organosilane (C): RS 3 (1.60 g) was converted intoethoxysilane (C) using the hydrosilylation procedure reported forpreparation of organosilane (8). The resultant crude organosilane waspurified by flash chromatography (silica, 5%acetonitrile/dichloromethane) to afford the purified organosilane (C)(1.03 g, 57.2% yield) as a yellowish solid. ¹ H NMR (CDCl₃)δ: 9.25(d,3H), 9.15 (d,6H), 4.6 (s,6H), 3.6 (q,2H), 1.8 (m,2H), 1.6 (m,2H), 1.3(m,12H), 0.5 (t,2H), 0.05 (s,6H).

Preparation of Recognition Stationary Phase RSP 3: Bonding of theorganosilane (C) to silica, and packing of the resulting stationaryphase into an HPLC column followed the procedure reported for thepreparation of stationary phase SP 5 (120° C., 1 mm Hg, 24 hours).Stationary phase recovered from the column packer was submitted forelemental analysis (C, 6.30%) which indicated a loading of 1.5×10⁻⁴moles of selector per gram of stationary phase.

PREPARATION OF RS 7

The synthetic route for RS 7 is shown in Table 2 supra.

Triol (B) (1.0 g) and triethylamine (2.2 g) were dissolved in 50 mldichloromethane and cooled in an ice bath. 2,4-dinitrofluorobenzene wasthen added. After 30 minutes the ice bath was removed and the reactionmixture was allowed to stir overnight at room temperature under nitrogenatmosphere. The crude reaction mixture was then evaporated and purifiedby flash chromatography on silica gel using dichloromethane as eluent togive RS 7 (1.39 g, 44% yield) as a pale yellow foam. ¹ H NMR (CDCl₃)δ:8.8 (d,3H), 8.5 (dd,3H), 7.35 (d,3H), 5.8 (m,1H), 4.95 (m,2H), 4.5(s,6H), 2.05 (m,2H), 1.85 (m,2H), 1.4 (m,12H).

Preparation of the Organosilane (D): RS 7 (1.39 g) was converted intoethoxysilane (D) using the hydrosilylation procedure reported forpreparation of organosilane (8). The resultant crude organosilane waspurified by flash chromatography on silica using 5% acetonitrile indichloro-methane as eluent to afford 1.0 g, the purified organosilane(D) (63% yield). ¹ H NMR (CDCL₃)δ: 8.8 (d,3H), 8.5 (dd,3H), 7.35 (d,3H),4.45 (s,6H), 3.65 (q,2H), 1.85 (m,2H), 1.25 (m,14H), 1.2 (t,3H), 0.55(t,2H), 0.1 (s,6H).

Preparation of Recognition Stationary Phase RSP 7: Bonding of theorganosilane (D) to silica, and packing of the resulting stationaryphase into an HPLC column followed the procedure reported for thepreparation of stationary phase 5 (130° C., 1 mm Hg, 24 hours).Stationary phase recovered from the column packer was submitted forelemental analysis (C, 6.29%) which indicated a loading of 1.6×10⁻⁴moles of selector per gram of stationary phase.

RESULTS AND DISCUSSION Evaluation of SP 1-8 and RSP 3 and RSP 7

Table 6 shows chromatographic data directed to the separation of theanalytes shown in Table 3, including and C₇₀, on SP's 1-8 and therecognition stationary phase of the present invention exemplified by RSP3 and RSP 7. As seen from Table 6, RSP 7 afforded the largest capacityfactor (k') for buckminsterfullerene (C₆₀).

                                      TABLE 6                                     __________________________________________________________________________    Capacity Factors for Stationary Phases SP 1-8 and RSP 3 and RSP 7             Analyte   SP 1                                                                             SP 2                                                                             SP 3                                                                             SP 4                                                                             SP 5                                                                             SP 6                                                                             SP 7                                                                             SP 8                                                                             RSP 3                                                                             RSP 7                                   __________________________________________________________________________    Benzene   0.18                                                                             0.15                                                                             0.11                                                                             0.12                                                                             0.12                                                                             0.13                                                                             0.10                                                                             0.10                                                                             0.14                                                                              0.17                                    Biphenyl  0.53                                                                             0.35                                                                             0.19                                                                             0.17                                                                             0.33                                                                             0.34                                                                             0.22                                                                             0.19                                                                             0.38                                                                              0.44                                    Naphthylene                                                                             0.77                                                                             0.46                                                                             0.19                                                                             0.19                                                                             0.40                                                                             0.44                                                                             0.26                                                                             0.27                                                                             0.58                                                                              0.58                                    Anthracene                                                                              2.19                                                                             1.06                                                                             0.34                                                                             0.30                                                                             1.12                                                                             1.24                                                                             0.70                                                                             0.70                                                                             1.74                                                                              1.28                                    Phenanthrene                                                                            2.57                                                                             1.19                                                                             0.34                                                                             0.31                                                                             1.22                                                                             1.35                                                                             0.72                                                                             0.81                                                                             2.06                                                                              1.41                                    1,2-Benzanthracene                                                                      5.55                                                                             2.27                                                                             0.57                                                                             0.47                                                                             3.00                                                                             3.31                                                                             1.97                                                                             1.88                                                                             5.61                                                                              3.30                                    Pyrene    5.96                                                                             2.27                                                                             0.43                                                                             0.39                                                                             2.53                                                                             2.73                                                                             1.49                                                                             2.11                                                                             5.84                                                                              2.68                                    Chrysene  6.30                                                                             2.43                                                                             0.60                                                                             0.50                                                                             3.23                                                                             3.46                                                                             2.09                                                                             2.13                                                                             6.45                                                                              3.57                                    C.sub.60  2.99                                                                             1.19                                                                             1.98                                                                             2.62                                                                             1.01                                                                             2.26                                                                             2.45                                                                             0.50                                                                             1.61                                                                              6.59                                    C.sub.70  4.65                                                                             1.90                                                                             4.16                                                                             5.31                                                                             1.80                                                                             4.76                                                                             7.10                                                                             0.80                                                                             3.28                                                                              20.77                                   α.sup.*                                                                           1.60                                                                             1.56                                                                             2.10                                                                             2.03                                                                             1.78                                                                             2.11                                                                             2.89                                                                             1.60                                                                             2.03                                                                              3.15                                    __________________________________________________________________________     Conditions: mobile phase = 5% dichloromethane in hexane, flow rate = 2.00     mL/min., ambient temperature, Void time determined using                      1,3,5tri-t-butylbenzene.                                                      *indicates separation factor for C.sub.60 and C.sub.70.                  

FIG. 3 depicts the relative capacity factors of the ten analytes testedon stationary phase SP 8 and RSP 3, which was formed with therecognition selector of the present invention. As seen in FIG. 3, thebest line through the data points for the polycyclic aromatichydrocarbon analytes had a slope of 3.03. The data points for thefullerene analytes C₆₀ and C₇₀ fell only slightly above the line definedby the polycyclic aromatic hydrocarbon analytes.

RSP 7, formed with the recognition selector of the present invention,afforded excellent retention and separation for all of the analytes. Ofthe stationary phases tested in Table 6, RSP 7 showed the greatestretention for the fullerenes and the best separation factor for the C₆₀/C₇₀ mixture. The relative capacity factors for all ten analytes on thetwo tripodal stationary phases of the invention that were tested, RSP 3and RSP 7, are illustrated in FIG. 4. It is believed that the reason RSP7 provided the degree of increased retention for the fullerenes relativeto the tripodal ester RSP 3 is the positioning of the π-acidic groups inRSP 7, which is one atom closer to the branching point than in RSP 3;this could conceivably impart some degree of conformational rigidity ora more favorable geometry for simultaneous multipoint interaction to RSP7.

What is claimed is:
 1. A method for separating a carbon cluster from a mixture containing the same which comprises contacting a mixture containing at least one carbon cluster with a recognition selector said recognition selector having the formula ##STR37## wherein R₂ is ##STR38## wherein R₂ is O, S or NR₁₂, wherein R₁₂ is independently hydrogen or P═O with the proviso that when R₁₂ is P═O, then only one such group is present and all R₂ 's are additionally bonded to R₁₂ ;R₄ is independently O, S or NH, R₃ and R₅ are each independently hydrogen or lower alkyl of up to 6 carbon atoms, n and o are each independently zero, 1, 2 or 3, p, q, r, s and t are each independently zero or 1, Ar is a monocyclic or ortho-fused polycyclic aromatic moiety having up to 10 carbon atoms, either of which may be unsubstituted or substituted with one or more lower alkyl of up to 6 carbon atoms, NO₂, N(R₆)₃ ⁺, CN, COOR₇, SO₃ H, COR₈ and OR₉ wherein R₆, R₇, R₈ and R₉ are each independently hydrogen or lower alkyl; W is H or CH═CH₂ ; and m is 1 to 10,under conditions effective to form a complex between said carbon cluster and said recognition selector; separating said complex from said mixture and recovering said carbon cluster from said complex.
 2. The method of claim 1 wherein said carbon cluster is C₆₀.
 3. The method of claim 1 wherein said carbon cluster is C₇₀.
 4. The method of claim 1, wherein in said recognition selectorn and p are each 1; o, q, r, s and t are each zero; R₂ is O; and Ar is 3,5-dinitrophenyl.
 5. The method of claim 1 wherein in said recognition selectorn and p are each 1; o, q, r, s and t are each zero; R₂ is NH; and Ar is 3,5-dinitrophenyl.
 6. The method of claim 1 wherein in said recognition selectorn, o, o and r are each 1; q, s and t are each zero; R₂ is O; R₄ is NH; and Ar is 3,5-dinitrophenyl.
 7. The method of claim 1 wherein in said recognition selectorn is 1; o, p, q, r, s and t are each zero; R₂ is O; and Ar is 2,4-dinitrophenyl.
 8. The method of claim 1 wherein in said recognition selectorn, p, q, r and s are each 1; o and t are each zero; R₂ and R₄ are each NH; R₃ is isobutyl; and Ar is 3,5-dinitrophenyl.
 9. The method of claim 1 wherein in said recognition selectorn, p, q and r are each 1; o, s and t are each zero; R₂ is O; R₃ is CH₃ ; R₄ is NH; and Ar is β-naphthyl.
 10. The method of claim 1 wherein in said recognition selectorn, p and q are each 1; o, r, s and t are each zero; R₂ is O; R₃ is CH₃ ; and Ar is 6-methoxy-β-naphthyl.
 11. The method of claim 1 wherein in said recognition selectorn, o, p, r, and t are each 1; q and s are each zero; R₂ is O; R₄ is NH; R₅ is CH₃ ; and Ar is α-naphthyl.
 12. The method of claim 1 wherein in said recognition selectorW is CH═CH₂ ; and M is
 7. 13. A method for separating carbon clusters having different numbers of carbon atoms which comprises contacting a mixture of at least one carbon cluster having a first number of carbon atoms and at least one carbon cluster having a second number of carbon atoms with a recognition selector, said recognition selector having the formula ##STR39## wherein R₁ is ##STR40## wherein R₂ is O, S or NR₁₂ wherein R₁₂ is independently hydrogen or P═O with the proviso that when R₁₂ is P═O, then only one such group is present and all R₃ 's are additionally bonded to R₁₂ ;R₄ is independently O, S or NH, R₃ and R₅ are each independently hydrogen or lower alkyl of up to 6 carbon atoms, n and o are each independently zero, 1, 2 or 3, p, q, r, s and t are each independently zero or 1, Ar is a monocyclic or ortho-fused polycyclic aromatic moiety having up to 10 carbon atoms, either of which may be unsubstituted or substituted with one or more lower alkyl of up to 6 carbon atoms, NO₂, N(R₆)₃ ⁺, CN, COOR₇, SO₃ H, COR₈ and OR₉ wherein R₆, R₇, R₈ and R₉ are each independently hydrogen or lower alkyl; W is H or CH═CH₂ ; and m is 1 to 10;under conditions effective to form a complex between said recognition selector and said carbon cluster having said first number of carbon atoms, and recovering said carbon cluster having said second number of carbon atoms.
 14. The method of claim 13 further comprising: subjecting the complex to conditions effective to dissociate said recognition selector from said carbon cluster having said first number of carbon atoms, and recovering said carbon cluster having said first number of carbon atoms.
 15. The method of claim 13 wherein a stationary phase in a chromatographic column comprises said recognition selector and the carbon cluster having said second number of carbon atoms elute from said column prior to said carbon cluster having said first number of carbon atoms.
 16. The method of claim 13 wherein said carbon cluster having said first number of carbon atoms is C₇₀ and said carbon cluster having said second number of carbon atoms is C₆₀.
 17. The method of claim 15 wherein said stationary phase has the formula ##STR41##
 18. The method of claim 15 wherein said stationary phase has the formula ##STR42##
 19. The method of claim 13 wherein a liquid membrane comprising said recognition selector is passed in contact with one side of a semi-permeable membrane and said mixture containing at least one carbon cluster having a first number of carbon atoms and at least one carbon cluster having a second number of carbon atoms is in contact with the other side of said semi-permeable membrane.
 20. A method for separating C₆₀ and C₇₀ which comprises passing a mixture containing at least one C₆₀ and at least one C₇₀ through a chromatographic column having a stationary phase of a recognition selector having the formula ##STR43## wherein R₁ is ##STR44## wherein R₂ is O, S or NR₁₂ wherein R₁₂ is independently hydrogen or P═O with the proviso that when R₁₂ is P═O, then only one such group is present and all R₂ 's are additionally bonded to R₁₂ ;R₄ is independently O, S or NH, R₃ and R₅ are each independently hydrogen or lower alkyl of up to 6 carbon atoms, n and o are each independently zero, 1, 2, or 3, p, q, r, s and t are each independently zero or 1, Ar is a monocyclic or ortho-fused polycyclic aromatic moiety having up to 10 carbon atoms, either of which may be unsubstituted or substituted with one or more lower alkyl of up to six carbon atoms, NO₂, N(R₆)₃ ⁺, CN, COOR₇, SO₃ H, COR₈ and OR₉ wherein R₆, R₇, R₈ and R₉ are each independently hydrogen or lower alkyl; W is H or CH═CH₂ ; and m is 1 to 10,immobilized on a support effective for use in chromatographic separation, under conditions effective to cause said C₆₀ to be less retained by said recognition selector than said C₇₀ thus causing said C₆₀ to elute from said column prior to said C₇₀.
 21. The method of claim 20 further comprising recovering said C₆₀ from said column.
 22. The method of claim 20 further comprising recovering said C₇₀ from said column.
 23. The method of claim 20 wherein said support is silica.
 24. The method of claim 23 wherein said stationary phase has the formula ##STR45##
 25. The method of claim 24 wherein said stationary phase has the formula ##STR46##
 26. A method for separating C₆₀ and C₇₀ which comprises passing a mixture containing at least one C₆₀ and at least one C₇₀ through a chromatographic column having a stationary phase of naphthylalanine or naphthylleucine immobilized on a silica or alumina support under conditions effective to cause said C₆₀ to be less retained by said stationary phase than said C₇₀ thus causing said C₆₀ to elute from said column prior to said C₇₀, said conditions effective including a temperature in the range of over room temperature to about 120° C.
 27. The method of claim 26 wherein said temperature is between about 80° C. and 100° C.
 28. The method of claim 27 wherein said temperature is about 90° C. 